Project Summary Bile acid signaling through FXR and TGR5 plays a critical role in the control of metabolism and inflammation in the liver. Accumulation of high levels of toxic bile acids causes liver inflammation and injury, contributing to the pathogenesis of chronic non-alcoholic fatty liver disease (NAFLD), diabetes and obesity. These inflammatory liver metabolic diseases have reached epidemic status in the U.S. population, and NAFLD occurs with a higher prevalence in males than females. A plethora of bile acid research in the last two decades has unveiled a complex network of pathways that integrate bile acid-activated farnesoid X receptor (FXR) and the bile acid- activated G protein-coupled receptor TGR5 signaling to regulate lipid, glucose, and energy metabolism and homeostasis. Bile acid synthesis is tightly regulated by a negative feedback mechanism to inhibit transcription of the gene encoding cholesterol 7?-hydroxylase (CYP7A1), the rate-limiting synthesis enzyme, and sterol 12?-hydroxylase (CYP8B1), required for cholic acid synthesis, in the classic bile acid synthesis pathway. The alternative pathway is regulated by oxysterol 7?-hydroxylase (CYP7B1). The gut microbiota regulates bile acid pool size, bile acid composition and enterohepatic circulation of bile acids. The anti-inflammatory action of bile acid-activated receptors has been recognized recently. However, the underlying molecular mechanisms of bile acid signaling in the regulation of hepatic metabolic homeostasis and inflammation are not fully understood. During the current funding period, we have used Cyp7a1-/-, Fxr-/- and Tgr5-/- mice to study the role of bile acid signaling in metabolic regulation. Activation of intestinal FXR reshaped the gut microbiota to activate TGR5, stimulating glucagon-like-peptide 1 (GLP-1) secretion, promoting white adipose tissue browning, and improving insulin sensitivity and glucose tolerance in obese and diabetic mice. We have successfully bred Fxr and Tgr5 double knockout (DKO) mice. DKO mice have increased bile acid synthesis and pool size and induction of the taurocholic acid-activated sphingosine-1-phosphate receptor 2 (S1PR2), the role of which in hepatic metabolism is not understood. Two specific aims are designed to 1) study the mechanisms of bile acid signaling in the regulation of hepatic bile acid synthesis and metabolic homeostasis, and 2) to study the role and mechanism of bile acid signaling in the pathogenesis of NAFLD. Metabolomics, 16S RNA-sequencing of the gut microbiome, and RNA-sequencing of the transcriptome will be used to study bile acid synthesis and hepatic metabolism in both male and female Fxr-/-, Tgr5-/- and DKO mice. This study is highly significant in elucidating the molecular mechanism of the regulation of bile acid synthesis and lipid homeostasis, and the mechanistic pathogenesis of liver-related metabolic diseases affecting a large population in the United States and worldwide.